Screening And Identification Of Differentially Expressed MRNA/lncRNA In GV/MII Oocytes

During the maturation of mammalian oocytes,many regulatory factors,cascade reactions and cell structure participate in regulation.With the application of single-cell sequencing technology,some IncRNA/mRNA that play an important role in the maturation of mammalian oocytes have been shown,but there are few studies in bovine oocytes.Therefore,this study used single-cell sequencing to analyze the differentially expressed transcripts and their enrichment pathways of GV and M? phases of bovine oocytes,screen candidate genes,and use mouse models for preliminary functional verification.In this study,GV and M? stage bovine oocytes were collected,single-cell sequencing technology and bioinformatics analysis were used to analyze the differential expression levels and enrichment pathways of oocytes at different stages,and to screen differentially expressed mRNA and lncRNA.Then,the genes Hdac8 and H2afz were selected and verified using a mouse model.Isolate 12.5d fetal mouse ovaries and 8d newborn mouse follicles,and then knock out Hdac8 or H2afz genes in vitro.The ovaries were transplanted under the capsulae renis for histological examination,the follicles were cultured in vitro and the diameter of the follicles was counted.The main results are as follows:(1)Sequencing of GV and M? oocytes obtained 124158786,170166366,197700530 and 215582682,214141570,188612824 raw readers,respectively.Remove low-quality sequences to obtain 105569888,149196588,174851682 and 190370022,183962404,166847838 clean readers.(2)After screening,6751 differentially expressed mRNAs were obtained,of which 3876 were significantly up-regulated in the M? phase,and 2875 were significantly down-regulated in the MiR phase.GO and KEGG functional enrichment analysis found that the significant enrichment includes the functional terms of cell metabolism process,oocyte maturation and cell cycle related signal pathways.(3)After screening,2744 differentially expressed lncRNAs were obtained,of which 1885 were significantly up-regulated in M? phase and 859 were significantly down-regulated in M? phase.GO and KEGG functional enrichment analysis found that the Cis target genes that differentially express lncRNA were significantly enriched in terms of biological synthesis process regulation function,cAMP signaling pathway,and cell cycle.Significantly enriched trans target genes include functional terms of cell metabolism and cell cycle related pathways.(4)The results of gene knockout ovaries showed that the follicles in the ovaries of the Hdac8-/-group developed slowly,mainly primary and secondary follicles.No follicles developed in the ovaries of the H2afz-/-group.Hoeever,in the control group,tertiary follicles were seen.(5)The results of gene knockout follicles showed that the diameter of follicles in the Hdac8-/-group increased during the first 5 days and decreased from the 6th day.The diameter of follicles in the H2afz-/-group increased during the first 4 days and decreased from the 5th day.In the control group,the diameter of the follicles kept increasing.In summary,this experiment reported for the first time the differentially expressed mRNAs and lncRNAs of bovine oocytes before and after maturation.These differentially expressed mRNAs and lncRNAs have significant differences in enrichment in lysine degradation,cAMP signaling,cell cycle,cell metabolism,and protein binding pathways.The candidate genes Hdac8 and H2afz play key roles in early follicle development.